Abstract
"Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (•OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved •OH footprinting is presented based on the Fenton reaction, Fe(II) + H2O2 → Fe(III) + •OH + OH-. It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 589-615 |
| Number of pages | 27 |
| Journal | Methods in Cell Biology |
| Volume | 84 |
| DOIs | |
| State | Published - 2008 |
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ASJC Scopus subject areas
- Cell Biology
Cite this
Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolution. / Shcherbakova, Inna; Mitra, Somdeb; Beer, Robert H.; Brenowitz, Michael D.
In: Methods in Cell Biology, Vol. 84, 2008, p. 589-615.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolution
AU - Shcherbakova, Inna
AU - Mitra, Somdeb
AU - Beer, Robert H.
AU - Brenowitz, Michael D.
PY - 2008
Y1 - 2008
N2 - "Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (•OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved •OH footprinting is presented based on the Fenton reaction, Fe(II) + H2O2 → Fe(III) + •OH + OH-. It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.
AB - "Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (•OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved •OH footprinting is presented based on the Fenton reaction, Fe(II) + H2O2 → Fe(III) + •OH + OH-. It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.
UR - http://www.scopus.com/inward/record.url?scp=35448978228&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=35448978228&partnerID=8YFLogxK
U2 - 10.1016/S0091-679X(07)84019-2
DO - 10.1016/S0091-679X(07)84019-2
M3 - Article
C2 - 17964944
AN - SCOPUS:35448978228
VL - 84
SP - 589
EP - 615
JO - Methods in Cell Biology
JF - Methods in Cell Biology
SN - 0091-679X
ER -